4.6 Article

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 33, Pages 23751-23764

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.483289

Keywords

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Funding

  1. Bundesministerium fur Bildung und Forschung (BMBF), Germany
  2. Netherlands Organization for Scientific Research (NWO), The Netherlands
  3. SysMo2
  4. European Union
  5. University of Groningen

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In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl) spermidine (T(SH)(2))). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 degrees C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays K-m values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 mu M, respectively, as well as K-i values for GSH and T(SH) 2 of 1 mM and 360 mu M, respectively. As Gsp hydrolysis has a K-m value of 5.6 mM, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)(2) inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

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